Algal elongase 6

ABSTRACT

Provided herein are exemplary isolated nucleotide sequences encoding polypeptides having elongase activity, which utilize fatty acids as substrates.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit and priority of U.S.Provisional Patent Application Ser. No. 61/480,364 filed on Apr. 28,2011, titled “Elongases,” which is hereby incorporated by reference.

The present application is related to U.S. Non-Provisional PatentApplication Ser. No. 12/581,812 filed on Oct. 19, 2009, titled“Homologous Recombination in an Algal Nuclear Genome,” which is herebyincorporated by reference.

The present application is related to U.S. Non-Provisional PatentApplication Ser. No. 12/480,635 filed on Jun. 8, 2009, titled “VCP-BasedVectors for Algal Cell Transformation,” which is hereby incorporated byreference.

The present application is related to U.S. Non-Provisional PatentApplication Ser. No. 12/480,611 filed on Jun. 8, 2009, titled“Transformation of Algal Cells,” which is hereby incorporated byreference.

REFERENCE TO SEQUENCE LISTINGS

The present application is filed with sequence listing(s) attachedhereto and incorporated by reference.

BACKGROUND OF THE INVENTION Field of the Invention

This invention relates to molecular biology, and more specifically, toalgal elongases.

SUMMARY OF THE INVENTION

Isolated nucleotide sequences encoding polypeptides having elongaseactivity, which utilize fatty acids as substrates.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the nucleotide sequence encoding elongase 1 (SEQ IDNO:1).

FIG. 2 illustrates the nucleotide sequence encoding elongase 2 (SEQ IDNO:2).

FIG. 3 illustrates the nucleotide sequence encoding elongase 3 (SEQ IDNO:3).

FIG. 4 illustrates the nucleotide sequence encoding elongase 4 (SEQ IDNO:4).

FIG. 5 illustrates the nucleotide sequence encoding elongase 5 (SEQ IDNO:5).

FIG. 6 illustrates the nucleotide sequence encoding elongase 6 (SEQ IDNO:6).

FIG. 7 illustrates the nucleotide sequence encoding elongase 7 (SEQ IDNO:7).

FIG. 8 illustrates the nucleotide sequence encoding elongase 8 (SEQ IDNO:8).

FIG. 9 illustrates the amino acid sequence encoding elongase 1 (SEQ IDNO:9).

FIG. 10 illustrates the amino acid sequence encoding elongase 2 (SEQ IDNO:10).

FIG. 11 illustrates the amino acid sequence encoding elongase 3 (SEQ IDNO:11).

FIG. 12 illustrates the amino acid sequence encoded by elongase 4 (SEQID NO:12).

FIG. 13 illustrates the amino acid sequence encoded by elongase 5 (SEQID NO:13).

FIG. 14 illustrates the amino acid sequence encoded by elongase 6 (SEQID NO:14).

FIG. 15 illustrates the amino acid sequence encoded by elongase 7 (SEQID NO:15).

FIG. 16 illustrates the amino acid sequence encoded by elongase 8 (SEQID NO:16).

DETAILED DESCRIPTION OF THE INVENTION

A fatty acid is a carboxylic acid with a long aliphatic tail (chain),which is either saturated or unsaturated. Saturated fatty acids arelong-chain carboxylic acids that usually have between 12 and 24 carbonatoms and have no double bonds. Unsaturated fatty acids have one or moredouble bonds between carbon atoms. Most naturally occurring fatty acidshave a chain of an even number of carbon atoms, from 4 to 28. Elongasesare enzymes which lengthen fatty acids by adding two carbon atoms to afatty acid's carboxylic acid end.

Provided herein are isolated nucleotide sequences encoding polypeptideshaving elongase activity, which utilize fatty acids as substrates.

The inventors sequenced the entire genome of algal genus Nannochloropsisand identified genes involved in fatty acid metabolism. They identifiedvarious elongases, including exemplary elongases which they designatedas elongases 1-9.

The inventors manipulated the activities of the above-specifiedexemplary elongase genes by:

1. Overexpression of the subject elongase gene with a strong promoter.

2. Promoter replacement or promoter insertion in front of the subjectelongase gene within the genome via homologous recombination.

3. Knock out of the subject elongase gene via insertion of atransformation construct into the gene or replacement of a part of orthe entire subject elongase gene via homologous recombination.

Exemplary support for the above-mentioned methods may be found in U.S.Non-Provisional Patent Application Ser. No. 12/581,812 filed on Oct. 19,2009, titled “Homologous Recombination in an Algal Nuclear Genome,” U.S.Non-Provisional Patent Application Ser. No. 12/480,635 filed on Jun. 8,2009, titled “VCP-Based Vectors for Algal Cell Transformation,” and U.S.Non-Provisional Patent Application Ser. No. 12/480,611 filed on Jun. 8,2009, titled “Transformation of Algal Cells,” all of which are herebyincorporated by reference.

Accordingly, the inventors were able to manipulate the activities of thevarious exemplary elongases for the purpose of modifying the contents ofcertain fatty acids within algal genus Nannochloropsis.

Some of these elongases, i.e. Elongases 6-8, are down-regulated underconditions when poly unsaturated fatty acid (“PUFA”) biosynthesis isdown-regulated as well (i.e. during Nitrogen starvation). These genesare excellent targets for over-expression, in order to achieve elevatedPUFA biosynthesis. Down-regulation of these (or other) genes, as anexample, by replacement of the endogenous promoter or insertion of aweaker promoter in front of the respective elongase gene could lead to ahigher content of short chain fatty acids. Down-regulation oftranscription could also be achieved, in some cases, by insertion of acommonly strong promoter in front of the respective elongase gene,presumably by modifying the respective chromatin arrangement around thesaid elongase gene, thus leading to a lower transcription level. Also,the introduction of point mutations into the gene when inserting anotherpromoter in front of such a gene via the homologous recombination flanksutilized, could lead to an altered activity of the respective geneproducts.

Over expression and knock out mutants of said elongase genes suggestthat at least 4 elongases with overlapping functions are operating inthe biosynthesis pathway leading to Eicosapentaenoic acid (“EPA”): theseare, but not limited to: Elongases 5, 6, 7, and 9. Transcriptomeanalysis also suggests that Elongase 8 is operating as well in the fattyacid biosynthesis pathway to EPA.

FIG. 1 illustrates the nucleotide sequence encoding elongase 1 (SEQ IDNO:1).

FIG. 2 illustrates the nucleotide sequence encoding elongase 2 (SEQ IDNO:2).

FIG. 3 illustrates the nucleotide sequence encoding elongase 3 (SEQ IDNO:3).

FIG. 4 illustrates the nucleotide sequence encoding elongase 4 (SEQ IDNO:4).

FIG. 5 illustrates the nucleotide sequence encoding elongase 5 (SEQ IDNO:5).

FIG. 6 illustrates the nucleotide sequence encoding elongase 6 (SEQ IDNO:6).

FIG. 7 illustrates the nucleotide sequence encoding elongase 7 (SEQ IDNO:7).

FIG. 8 illustrates the nucleotide sequence encoding elongase 8 (SEQ IDNO:8).

FIG. 9 illustrates the amino acid sequence encoding elongase 1 (SEQ IDNO:9).

FIG. 10 illustrates the amino acid sequence encoding elongase 2 (SEQ IDNO:10).

FIG. 11 illustrates the amino acid sequence encoding elongase 3 (SEQ IDNO:11).

FIG. 12 illustrates the amino acid sequence encoded by elongase 4 (SEQID NO:12).

FIG. 13 illustrates the amino acid sequence encoded by elongase 5 (SEQID NO:13).

FIG. 14 illustrates the amino acid sequence encoded by elongase 6 (SEQID NO:14).

FIG. 15 illustrates the amino acid sequence encoded by elongase 7 (SEQID NO:15).

FIG. 16 illustrates the amino acid sequence encoded by elongase 8 (SEQID NO:16).

While various embodiments have been described above, it should beunderstood that they have been presented by way of example only, and notlimitation. Thus, the breadth and scope of a preferred embodiment shouldnot be limited by any of the above-described exemplary embodiments.

1-16. (canceled)
 17. A vector comprising a polynucleotide sequenceencoding elongase 6 having the amino acid sequence of SEQ ID NO:14. 18.The vector of claim 17, wherein the polynucleotide sequence is SEQ IDNO:6.
 19. The vector of claim 17, further comprising aViolaxanthin-chlorophyll a binding protein (VCP) promoter.
 20. Thevector of claim 17, which is a transformation vector.
 21. The vector ofclaim 17, which is a homologous recombination vector.
 22. The vector ofclaim 17, which has been incorporated into the genome of an algal cell.23. A method of modulating poly unsaturated fatty acid biosynthesis inan algal cell, the method comprising enhancing or suppressing theexpression of elongase 6 having the amino acid sequence of SEQ ID NO:14.24. The method of claim 23, wherein the expression of elongase 6 isenhanced and EPA production is increased in the algal cell.
 25. Themethod of claim 23, wherein the expression of elongase 6 is suppressedand EPA production is decreased in the algal cell.
 26. The method ofclaim 23, wherein the algal cell is a Nannochloropsis cell.
 27. Themethod of claim 24, wherein the algal cell is present in aNannochloropsis alga.
 28. The method of claim 23, wherein the algal cellhas been transformed with a stronger or weaker promoter in front of agenomic sequence encoding elongase 6 having the amino acid sequence ofSEQ ID NO:14.
 29. The method of claim 25, wherein the algal cell hasbeen transformed to result in (1) an insertion in the genomic sequenceencoding elongase 6 having the amino acid sequence of SEQ ID NO:14; or(2) a deletion or substitution of a portion or the full length of thegenomic sequence encoding elongase
 6. 30. A transformed algal cell withaltered poly unsaturated fatty acid biosynthesis, wherein the algal cellhas altered expression level of elongase 6 having the amino acidsequence of SEQ ID NO:14.
 31. The algal cell of claim 30, wherein astronger or weaker promoter is placed in front of a genomic sequenceencoding elongase 6 having the amino acid sequence of SEQ ID NO:14. 32.The algal cell of claim 30, which has (1) an insertion in the genomicsequence encoding elongase 6 having the amino acid sequence of SEQ IDNO:14; or (2) a deletion or substitution of a portion or the full lengthof the genomic sequence encoding elongase
 6. 33. The algal cell of claim30, which is a Nannochloropsis cell.
 34. The algal cell of claim 33,which is a part of a Nannochloropsis alga.